Acetyl-CoA carboxylase (ACC) catalyzes the first step in fatty acid biosynthesis and its product, malonyl-CoA, inhibits fatty acid oxidation. These functions make ACC a prime target for the development of therapeutics to treat obesity and type 2 diabetes. Knockout studies in mice, and animal studies with ACC inhibitors, have validated this approach. The most potent inhibitor of eukaryotic ACCs identified to date is the natural product soraphen. Soraphen has been demonstrated to have pharmacological properties consistent with the potential to treat obesity and diabetes; however, soraphen is not suitable as a therapeutic due to toxic side effects. Furthermore, soraphen is not a good starting point for drug development due to its complex structure. We propose to identify novel small molecules that target the soraphen binding site and inhibit mammalian ACCs with high potency. In Phase I research we cloned and expressed biotin carboxylase (BC) domains from the human ACC1 and ACC2 isozymes that retain high affinity soraphen binding, developed a high throughput screening assay, and solved the x-ray crystal structure for yeast BC alone and in complex with soraphen. In Phase II research we will use these tools to identify novel inhibitors by completing the following specific aims: 1) molecular docking of approximately 2,000,000 commercially available compounds to the soraphen binding site; 2) high throughput screening of the top ranked docking hits, and of an in- house small molecule library; 3) in vitro and in vivo characterization of leads; 4) lead optimization through iterative synthesis and testing of analogs; 5) determination of the structures of additional BC domains and of novel leads in complex with the BC domain; and 6) efficacy studies in animals with 1 to 3 leads to demonstrate potential for treating obesity and type 2 diabetes. This project will discover new drug candidates for combating obesity and diabetes. Combined, these diseases affect approximately 35% of Americans and cost approximately $200 billion annually. [unreadable] [unreadable] [unreadable]